FLUORESCENCE PULSE SHAPE AS A MORPHOLOGICAL INDICATOR IN THE ANALYSIS OF COLONIAL MICROALGAE BY FLOW-CYTOMETRY

Individual algal cells and colonies were entrained in a fluid stream and passed through a tightly focused (200 × 10 μm) laser beam to generate chlorophyll-fluorescence pulses. Simple pulse-shape parameters (duration, height and integral) were extracted by analogue circuitry and the relationship between these parameters analysed digitaly. Pulse shape analysed in this way provided low-resolution morphological information at a throughput rate up to 1000 cells·s-1: such information is particularly useful in the flow analysis of larger algal cells and colonial species. Successful implementation of the technique required that the flow regime oriented the cells uniformly and stably in the laser-beam waist and it worked best with a wide-bore (300 μm) hydrodynamic focusing nozzle which resisted clogging and avoided disrupting colonies by shearing. As a demonstration of feasibility, the technique was used to measure the length distribution of filaments of the cyanobacterium Anabaena solitaria and to discriminate between these filaments and globular colonies of the green alga Eudorina unicocca. Since a low power (100 mW) Ar laser was found to provide adequate illumination, the method can be implemented using low-cost cytometric equipment. © 1990.